SUMMARY/ABSTRACT The key obstacle to cure HIV infection is a reservoir of latently-infected memory CD4+ T-cells that persist despite long-term ART and cause a rebound of viremia if therapy is interrupted. This cellular reservoir has been shown to include mostly central memory, transitional memory, and stem cell memory CD4+ T-cells. Several studies have suggested that homeostatic proliferation of latently-infected memory CD4+ T-cells plays a key role in maintaining the overall size of the reservoir. Collectively, these data suggest that long-lived memory CD4+ T-cells with enhanced self-renewal capacity such as the CD4+ TSCM and TCM represent a major obstacle to HIV eradication and that clinical interventions aimed at curing HIV should be designed to specifically target the proliferation of these cell populations. In our lab, we recently evaluated a strategy to target the self-renewal capacity of memory CD4+ T-cells through the pharmacological inhibition of the Wnt signaling pathway in SIV-infected, ART-suppressed macaques. Wnt inhibition with PRI-724 induced TSCM and TCM differentiation as evidenced by the cell gene expression profiles following treatment. Furthermore, a decline in SIV DNA levels following PRI-724 treatment was observed in the lymph nodes (LN) TCM and TFH CD4+ T-cells. Here, we propose to target simultaneously the two main signaling pathways regulating memory T-cell proliferation during the critical period of reservoir establishment. We hypothesize that combined inhibition of the Wnt and Notch signaling pathways during acute SIV infection will block the self-renewal of long-lived memory CD4+ T-cells thus altering the stability of the viral reservoir over time. This hypothesis will be addressed by the experiments in the following Specific Aims: Aim 1. To establish a robust regimen of Wnt and Notch inhibitors to maximize the blockade of CD4+ TSCM and TCM self-renewal in healthy macaques. Aim 2. To assess in vivo the effect of Wnt and Notch combined inhibition during acute SIV infection on viral reservoir establishment in macaques. The proposed studies build upon an appreciation of the immunologic complexity of the CD4+ T-cell reservoir. This conceptually innovative work will provide critical new information on the biology of HIV/SIV persistence in memory CD4+ T-cells. The results of this project may lead to a translational research program focusing on novel CD4+ TSCM- and TCM-specific strategies for HIV eradication with the potential to significantly impact HIV treatment and cure approaches.